The isolated, perfused rat liver will be used in a continuation of studies on protein turnover and its hormonal and non-hormonal regulation. Studies completed thus far have shown that the release of C14 valine from previously labeled perfused livers into a large pool of carrier valine (15 mM) provides a valid measurement of overall proteolysis. This process increases spontaneously during control perfusion and with the administration of glucagon and is inhibited by insulin, amino acids and cycloheximide. Analyses of perfused liver tissue has revealed alterations of proteolytic activity in mitochondrial plus lysosomal fractions and changes in the sensitivity of lysosomes to osmotic shock which correlate positively with rates of proteolysis and appear to account for a large fraction of the proteolytic activity in the intact liver. Studies are planned to test the notion that lysosomal alterations induced by hormonal and non-hormonal treatment during perfusion are largely responsible for the observed changes in liver protein turnover. Lysosomes will be fractionated by equilibrium density centrifugation for the evaluation of their physical properties and protein content. Quantitative comparisons will be made between proteolytic rates in the intact perfused liver and in fractions of tissue homogenates. Further studies are planned to assess the importance of the regulation of hepatic proteolysis in the maintenance of free amino acid pools and in the repletion of hepatic protein reserves.